1. Field of the Invention
The present invention relates to a hydroxy carboxylic ester asymmetric hydrolase (Hydroxy Carboxylic ester Hydrolase: hereinafter abbreviated to as “EnHCH”), a gene encoding the same, a recombinant vector containing the gene, a transformant transformed by the recombinant vector and a process for producing the enzyme using the transformant, which EnHCH is a biological catalyst useful for the preparation of an optically active chlorohydrin, optically active 3-hydroxy-γ-butyrolactone, optically active hydroxycarboxylic acid, and its antipode alkyl ester which is a useful chiral building block in the synthesis of an optically active compound to be used for medicines, agricultural chemicals, and strongly dielectic liquid crystal, etc. Moreover, it relates to a process for the preparation of an optically active material using the enzyme and the transformant.
2. Prior Art
Optically active compounds have been usually produced by a chemical synthesis comprising converting the corresponding optically active starting compound into the desired compound, or by an optical resolution comprising treating the corresponding racemic compound with an optically resolving agent, but recently, it is reported to produce the optically active compound by a biological optical resolution utilizing an asymmetric reduction or asymmetric hydrolysis of a racemic compound with a microorganism or an enzyme.
As a method of preparing optically active 4-chloro-3-hydroxycarboxylic ester, E. Santaniello, et al. have reported a process for prepaing S-ethyl 4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate by asymmetric reduction using baker's yeast (E. Santaniello et al., Journal of Chemical Research (J. Chem. Research), 1984, pp. 132–133). Also, Takahashi et al. have reported a process for the preparation of optically active ethyl 4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate by asymmetric reduction using microorganisms (Japanese Laid-Open Patent Application No. Sho. 61–146191).
As a preparation process using an enzyme, Peters et al. have reported a process for the preparation of S-methyl 3-hydroxybutanoate and R-ethyl 4-chloro-3-hydroxybutanoate from methyl 3-oxobutanoate or ethyl 4-chloro-3-oxobutanoate by asymmetric reduction using carbonyl reductase for Rhodococcus erythropolis (J. Peters et al., Applied Microbiology Biotechnology (Appl. Microbiol. Biotechnol.), 1992, vol. 38, pp. 334–340, T. Zelinski et al., Journal of Biotechnology (J. Biotechnol.), 1994, vol. 33, pp. 283–292). Moreover, Shimizu et al. have reported a preparation process of R-ethyl 4-chloro-3-hydroxybutanoate by asymmetric reduction using aldehyde reductase for Sporoboromyces salmonicolor AKU4429 strain (Shimizu et al., Biotechnology Letter (Biotechnol. Lett.), 1990, vol. 12, pp. 593–596, Shimizu et al., Applied Microbiology Biotechnology (Appl. Microbiol. Biotechnol.), 1990, vol. 56, pp. 2374–2377).
However, in the preparation method of an optically active β-hydroxy ester compound from a prochiral β-keto ester compound by asymmetric reduction using these microorganisms or enzymes, an expensive coenzyme such as NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate), etc. are required for the reaction, and its oxidized product is required to be converted again into a reduced material whereby an enzyme such as glucose oxidase or formic acid dehydrogenase, etc. is separately required. Moreover, the above reaction step becomes a rate-determining reaction, and thus, the above-mentioned process cannot be said to be an industrially useful process.
As a process for the preparation of optically active 3-hydroxybutanoate, it has been known a process for the preparation of S-3-hydroxybutanoate from acetoacetate by asymmetric reduction using microorganisms such as yeast (Hamdani et al., Tetrahedron: Asymmetery, 1991, vol. 2, pp. 867–870) or Halobacterium halobium, (Ehrler and Seebach, Helvetica Chimica Acta (Helv. Chim. Acta), 1989, vol. 72, pp. 793–799). However, in this method, an expensive coenzyme such as NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate), etc. are required for the reaction.
When the above-mentioned Halobacterium halobium is acted on racemic 3-hydroxybutanoate, it has been reported that R-3-hydroxybutanoate remains by carboxylate hydrolysis reaction. It has been not known, however, a process for the preparation of S-3-hydroxybutanoate with high optical purity from racemic 3-hydroxybutanoate using a microorganism having a stereospecific ester resolution activity.
As a process for the preparation of optically active 2-hydroxybutanoate, it has been known a process from 2-hydroxyhexadecanoic ester or 2-hydroxytetracosanoic ester by an interesterification reaction of a secondary alcohol using lipase (Sugai et al., Yukigosei Kagakukaishi (Journal of Organic Syntesis Chemistry Association), 1995, vol. 53, pp. 48–58). However, it has been not known a process for the preparation of optically active 2-hydroxybutanoate by hydrolysis reaction of a carboxylate.
As a process for the preparation of optically active lactic acid utilizing microorganisms or enzymes, there have been known a fermentation method from glucose using lactic acid bacterium (Brin, Biochemical Preparation (Biochem. Prepn.), 1953, vol. 3, p. 61; Andersen and Greaves, Industrial Engineering Chemistry (Ind. Eng. Chem.), 1942, vol. 34, p. 34) or a preparation process from 2-halopropionic acid by a dehalogenation enzyme using a Pseudomonas genus microorganism (Soda et al., Biodegradation, 1995, vol. 6, pp. 223–227). However, it has been not known a process for the preparation of optically active lactate or lactic acid by stereospecifically hydrolyzing a carboxylate.
As a process for the preparation of optically active tetrahydrofuran-2-carboxylate by utilizing an enzyme, it has been known a process for the preparation of the same from a racemic mixture by stereospecific hydrolysis using various kinds of protease, lipase (WO 01/92553-A, WO 01/92554-A), esterase (Japanese Laid-Open Patent Application No. 2002-171994) each derived from nature. However, there is no process which can obtain R-methyl tetrahydrofuran-2-carboxylate with high optical purity. Also, the above process requires an expensive enzyme.
By the reasons as stated above, it has been strongly desired to culture a separated strain, to produce an enzyme with high properties with an inexpensive cost and a large amount whereby applying the enzyme to a process for the preparation of optically active hydroxycarboxylic acid and its antipode alkyl ester to make the process simple and ease.
Also, if a gene of the enzyme can be subjected to cloning, it is possible to produce the enzyme with an inexpensive cost and a large amount by using a genetic engineering technique, so that is has been strongly desired to subject the gene encoding the enzyme to cloning.
As a process for solving the above problems, the present inventors have already found Enterobacter sp. DS-S-75 strain (FERM BP-5494) separated from soil which is a bacterium belonging to the genus Enterobacter and having an activity of subjecting to stereoselective ester hydrolysis reaction and proposed a process for converting S-4-chloro-3-hydroxybutanoate into optically active 3-hydroxy-γ-butyrolactone by acting microorganisms or a product thereof on a chlorohydrin compound such as racemic 4-chloro-3-hydroxybutanoate to effect stereoselective dechlorination and ester hydrolysis reaction, and to recover a remaining another R-4-chloro-3-hydroxybutanoate simultaneously (see Japanese Laid-Open Patent Application No. Hei. 9-47296 and U.S. Pat. No. 5,776,766, Suzuki et al., Enzyme and Microbial Technology, 19.99, vol. 24, pp. 13–20).
They have further studied about a substrate specificity in the above-mentioned microorganism raction, and as a result, they have found that the microorganisms have characteristics of stereoselectively degrading various kinds of hydroxycarboxylic esters (Japanese Patent Application No. Hei. 13-391726). They have further studied, and as a result, they have succeeded in purifying asymmetric hydrolase (EnHCH) which participates in the above-mentioned microorganism reaction from the cells of the microorganism and in obtaining a EnHCH gene encoding the same.